EP0069281B1 - Elément d'analyse à plusieurs couches; procédé pour sa préparation et son emploi dans des procédés analytiques - Google Patents

Elément d'analyse à plusieurs couches; procédé pour sa préparation et son emploi dans des procédés analytiques Download PDF

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EP0069281B1
EP0069281B1 EP82105549A EP82105549A EP0069281B1 EP 0069281 B1 EP0069281 B1 EP 0069281B1 EP 82105549 A EP82105549 A EP 82105549A EP 82105549 A EP82105549 A EP 82105549A EP 0069281 B1 EP0069281 B1 EP 0069281B1
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Prior art keywords
ligand
layer
reagents
reagent
reagent layer
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EP0069281A1 (fr
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Bert Walter
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Bayer Corp
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Miles Inc
Miles Laboratories Inc
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
    • G01N33/525—Multi-layer analytical elements
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/805—Test papers
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/807—Apparatus included in process claim, e.g. physical support structures
    • Y10S436/81—Tube, bottle, or dipstick

Definitions

  • the present invention relates to the field of analytical test elements and methods, such as are useful in manual and automated diagnostic systems, and, more particularly, to multilayer analytical elements useful in the qualitative and quantitative determination of body fluid constituents and medicaments present in such body fluids.
  • Test devices in the form of test strips and similar solid stage analytical elements have become commonplace in the analysis of various types of samples, particularly biological fluids.
  • Test strips designed for detecting clinically significant substances in biological fluids, such as serum and urine, have been advantageous in the diagnosis of disease.
  • Test strips of various types have been known and used for many years in a wide variety of fields, from the most familiar pH test paper devices to in vitro diagnostic devices for the detection of various urine and blood components such as glucose, protein, occult blood and so forth (e.g., as described in U.S. Patent Nos. 3,164,534; 3,485,587; and 3,012,976).
  • Reagent compositions found in such test strips often having limited sensitivity, interact with the constituent or constituents to be determined by direct chemical reaction and are applied to the detection of substances that are present in liquid samples at concentrations in the millimolar range or above.
  • a basic multilayer integral analytical element is described in U.S. Patent No. 3,092,465.
  • Such multilayer elements use an absorbent fibrous carrier impregnated with one or more reagents, typically including a color former, over which is coated a semi-permeable membrane.
  • reagents typically including a color former
  • a semi-permeable membrane Upon contact with a test liquid, analyte passes through the membrane and into the fibrous carrier to generate color in an amount related to the concentration of analyte.
  • the membrane prevents passage and absorption of certain interfering components such as red blood cells, that could impair accurate reading of the color provided as a test result.
  • multilayer integral analytical elements are described in U.S. Patent No. 3,992,158. Such elements can receive a liquid sample and spread the sample within a spreading layer of the element to obtain in the element an apparent uniform concentration of analyte, other appropriate sample constituent or analyte product and produce in the presence of analyte an analytical result that can be measured quantitatively by automated devices, using techniques such as spectrophotometry, fluorimetry, etc.
  • Such elements can include spreading layers and reagent layers that contain a reactive or otherwise interactive material that, by virtue of its activity, promotes in the element a radiometrically detectable change, such as a color change.
  • U.S. Patent No. 4,042,335 relates to an element having (1) a reagent layer which reacts with the analyte to form a diffusible, detectable species; (2) a nonfibrous radiation blocking layer, permeable to the detectable species and having an opacifying agent; and (3) a non-fibrous, radiation-transmissive registration layer in which the detectable species is detected.
  • the element is, thus, read from below.
  • U.S. Patent No. 4,066,403 (Re 30,267) relates to an element including (1) a reagent which reacts with the analyte to produce a decomposition product; and (2) a reagent which reacts with the decomposition product or an intermediate to provide a detectable change, and having, as an improvement, a barrier composition separating reagent (1) from reagent (2), and being substantially uniformly permeable to the decomposition product and substantially impermeable to interferants. Therefore, what this does is add a "filtering" layer between the "reagent" layer and the "registration" layer.
  • U.S. Patent No. 4,144,306 relates to an element in which the reagent layer contains a nondiffusible material including a preformed, detectable moiety which is released and becomes diffusible in the presence of the analyte.
  • the registration layer receives the diffusible species.
  • Layers within the element are composed such that the preformed, detectable moiety released from the reagent layer can be detected selectively within the element.
  • U.S. Patent No. 4,166,093 relates to an element having (1) a radiation-transmissive reagent layer that reacts with an analyte to provide a detectable species, and (2) a porous radiation-blocking layer which is permeable to the analyte. As an improvement, it also has (3) a radiation-transmissive, detectable species migration-inhibiting layer between the reagent layer and the porous radiation-blocking layer. The migration-inhibiting layer is permeable to the analyte and inhibits the migration of the detectable species to the radiation-blocking layer.
  • Solid phase test devices have been applied to heterogeneous specific binding assays in attempts to overcome the inconveniences and disadvantages of the requisite separation step.
  • a commonly used solid phase device of this type comprises a non-porous surface, such as the interior surface of a test tube or other vessel, to which antibody is affixed or coated by adsorption or covalent coupling.
  • U.S. Patent Nos. 3,826,619; 4,001,583; 4,017,597; and 4,105,410 relate to the use of antibody coated test tubes in radioimmunoassays.
  • Solid phase test devices have also been used in heterogeneous enzyme immunoassays (U.S. Patent Nos. 4,016,043 and 4,147,752) and in heterogeneous fluorescent immunoassays (U.S. Patent Nos. 4,025,310 and 4,056,724; and British Patent Spec. No. 1,552,374).
  • test device is incorporated with the antibody reagent and is brought into contact with the liquid sample and with remaining reagents of the reaction system, principally the label conjugate. After an incubation period, the solid phase device is physically removed from the reaction solution and the label is measured either in the solution or on the test device.
  • heterogeneous specific binding assay test devices have been described wherein most or all of the necessary reagents are incorporated with the same carrier element, and wherein reagent/sample contacts and separation of the free- and bound-phases are accomplished by capillary migrations along the carrier element (U.S. Patent Nos. 3,641,235; 4,094,647 and 4,168,146).
  • the devices described in such patents are generally considered difficult to manufacture and susceptible to irreproducibility due to the complex nature of the many chemical and physical interactions that take place along the carrier element during performance of an assay.
  • Yet another approach to a heterogeneous immunoassay element is exemplified by European Patent Application 0 013 156.
  • British Patent No. 1,552,607 describes homogeneous specific binding assay systems employing various novel labels, including chemi- luminescent labels, enzyme substrate labels and coenzyme labels.
  • various novel labels including chemi- luminescent labels, enzyme substrate labels and coenzyme labels.
  • line 12 et seq of this patent there is the suggestion to incorporate the assay reagents with various carriers including liquid-holding vessels or insoluble, porous, and preferably absorbent, matrices, fleeces, or flocks; gels; and the like. This lacks a detailed teaching of how to apply homogeneous specific binding assay reagent systems to solid state test devices.
  • DE-A-2,537,275 describes a homogeneous specific binding assay reagent system and poses the possibility of using slides or strips incorporated with antibody in performing the assay.
  • the label conjugate would first be mixed wihh the sample and thereafter the antibody incorporated test device contacted with the reaction mixture. After a suitable incubation time, it is proposed that the test device would be rinsed with buffer, dried, and then the signal (fluorescence) measured.
  • this publication poses a test device and assay method much like those already known for heterogeneous specific binding assay techniques wherein the test device is immersed in the liquid reaction mixture, incubated, thereafter removed, washed, and finally read.
  • the proposed test device does not incorporate all of the binding assay reagents with the carrier element. Specifically, only the antibody is proposed to be incorporated with the carrier element, the label conjugate being separately added to the sample under assay prior to contact with the proposed test device.
  • Blocking layers used in prior art elements are required to be permeable to the ligand, reagents of the reagent layer, or products of their interreaction since the response of the element is read from the element surface away from the reagent layer, i.e., the support layer surface.
  • the problem is that electromagnetic radiation, such as emitted in reflectance and fluorescence systems, is affected by support layers, such as polystyrene or polyester layers, through which it must pass in these prior art elements. A portion of the electromagnetic radiation, such as light, which passes through the support layer is trapped inside the layer. Thus, it acts, in essence, as a fiber optic. As such, the amount of electromagnetic radiation, such as light, which is detected does not precisely indicate the amount resulting from the reaction which has occurred in the element. Dose response results and the like therefore do not entirely represent the amount of electromagnetic radiation from the response to the ligand in the reagent layer.
  • the element of the invention emits an enhanced electromagnetic response, or signal, as compared to prior art elements. Even more notably, the ratio of signal radiation (S) to background radiation (B) emitted is enhanced, in that interfering background radiation is avoided, by a factor of at least an order of magnitude (10x), as is demonstrated in the examples, infra.
  • a multilayer analytical element for detecting a ligand in or the ligand binding capacity of a liquid sample of the type having a reagent layer incorporating reagents which are responsive to the ligand in or the ligand binding capacity of the sample to give a detectable response, a radiation diffusing and blocking layer, and a support layer, characterized in that the radiation diffusing and blocking layer is (a) interposed between the reagent layer and the support layer; (b) impermeable to the ligand, reagents of the reagent layer, and products of their interreaction; (c) inert to the ligand, reagents present in the reagent layer, and products of their interreaction; (d) between 0.0005 and 0.05 cm in thickness; and (e) opaque and contains a radiation impermeable pigment.
  • ligand is used to refer to body fluid constituents and medicaments or other substances present in such body fluids. The following exemplifies a number of such possible ligands.
  • Reagent compositions are known for blood, plasma or serum ligands such as ascorbic acid, bile acids bilirubin, cholesterol, creatinine, glucose, lactic acid, phospholipids, triglycerides, urea nitrogen (BUN) and uric acid.
  • serum ligands such as ascorbic acid, bile acids bilirubin, cholesterol, creatinine, glucose, lactic acid, phospholipids, triglycerides, urea nitrogen (BUN) and uric acid.
  • blood chemistry enzyme ligands such as amylase, cholinesterase, creatine phosphokinase (CPK), the dehydrogenases (hydroxybutyric, isocitric, lactic and malic acids), lipase, phenylalanine, the transaminases (glutamic oxaloacetic and glutamic pyruvic), acid and alkaline phosphatases, gamma-glutamyl transpeptidase, leucine aminopeptidase and the erythrocyte enzymes (glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, glutathione reductase and pyrubate kinase).
  • ligands such as amylase, cholinesterase, creatine phosphokinase (CPK), the dehydrogenases (hydroxybutyric, isocitric, lactic and malic acids), lipase, phenylalanine, the transa
  • blood protein ligands such as albumin, cryoglobins, components of the coagulation and fibrinolytic systems, complement factors and the cellular and serum immune effectors such as interferon and immunoglobins, as is more fully discussed, infra, with reference to homogeneous specific binding assays.
  • reagent compositions are known for urine chemistry determinations.
  • such ligands generally include ascorbic acid, albumin, creatine, creatinine, glucose, bile acids, bilirubin, protein, ketones, occult blood, nitrite, amylase and phenylpyruvic acid.
  • the present assay element may also be applied to the detection of any ligand for which there is a specific binding partner and, conversely, to the detection of the capacity of a liquid medium to bind a ligand (usually due to the presence of a binding partnerforthe ligand in the medium).
  • the ligand usually is a peptide, polypeptide, protein, carbohydrate, glycoprotein, steroid, or other organic molecule for which a specific binding partner exists in biological systems or can be synthesized.
  • the ligand in functional terms, is usually selected from antigens and antibodies thereto; haptens and antibodies thereto; and hormones, vitamins, metabolites and pharmacological agents, and their receptors and binding substances.
  • the ligand is an immunologically-active polypeptide or protein or molecular weight between 1,000 and 10,000,000, such as an antibody or antigenic polypeptide or protein, or a hapten of molecular weight between 100 and 1,500.
  • polypeptide ligands are angiotensin I and II, C-peptide, oxytocin, vasopressin, neurophysin, gastrin, secretin, bradykinnin, and glucagon.
  • Representative protein ligands include the classes of protamines, mucoproteins, glyco- ptcteins, globulins, albumins, scleroproteins, phosphoproteins, histones, lipoproteins, chromo- proteins, and nucleoproteins.
  • proteins examples include prealbumin, a,-lipoprotein, human serum albumin, a,-glycoprotein, transcortin, thyroxine binding globulin, hapto- globin, hemoglobin, myoglobin, ceruloplasmin, o2-lipoprotein, a 2 -macroglobulin, ⁇ -lipoprotein, erythropoietin, transferrin, homopexin, fibrinogen, the immunoglobulins such as IgG, IgM, IgA, IgD, and IgE, and their fragments, e.g., F c and F ab , complement factors, prolactin, blood clotting factors such as fibrinogen, thrombin and so forth, insulin, melanotropin, somatotropin, thyrotropin, follicle stimulated hormone, leutiniz- ing hormone, gonadotropin, thyroid stimulating hormone, placental lactogen, intrinsic factor, transcobalamin
  • hapten ligands include the general classes of drugs, metabolites, hormones and vitamins.
  • Haptenic hormones include thyroxine and triiodothyronine.
  • Vitamins include vitamins A, B, e.g., B 12 , C, D, E and K, folic acid and thiamine.
  • Drugs include antibiotics such as aminoglycosides, e.g., gentamicin, tobramycin, amikacin, sisomicin, kanamycin, and netilmicin, penicillin, tetracycline, terramycin, chloro- mycetin, and actinomycetin; nucleosides and nucleotides such as adenosine diphosphate (ADP), adenosine triphosphate (ATP), flavin mononucleotide (FMN), nicotinamide adenine dinucleotide (NAD) and its phosphate derivative (NADP), thymidine, guanosine and adenosine; prostaglandins; steroids such as the estrogens, e.g., estriol and estradiol, st6rogens, androgens, digoxin, digitoxin, and adrenocortical steriods; and others such as phenobarbital
  • the liquid medium to be assayed can be a naturally occurring or artificially formed liquid suspected to contain the ligand, and usually is a biological fluid or a dilution thereof.
  • Biological fluids that can be assayed include serum, plasma, urine, saliva, and amniotic and cerebrospinal fluids.
  • the position of the radiation diffusing and blocking layer, relative to the reagent layer(s) and support layer, is critical to the present invention.
  • the characteristics of the radiation diffusing and blocking layer arise from the constituents with which it is prepared and these characteristics are, likewise, critical to the invention.
  • the radiation blocking layer of the invention is a radiation diffusing and blocking layer which is interposed between the reagent layer(s) and the support layer. It can be direct contact with one surface of the reagent layer(s); the signal emitted from the reagent layer is then read from the other surface of the reagent layer(s). As such, the radiation signal is not required to pass through any layer or material for detection other than the reagent layer from which it is emitted.
  • suitable treatment e.g., treatment with sodium hydroxide
  • a subbing layer can be positioned between the radiation diffusing and blocking layer and the reagent layer(s) to improve the bonding or adherence of the reagent layer.
  • the characteristics or properties of the radiation diffusing and blocking layer are that it is opaque, relatively thin and both impermeable and inert to the ligand, reagents of the reagent layer, and products of their interreaction.
  • the radiation diffusing and blocking layer contains a radiation impermeable pigment, preferably uniformily distributed throughout the layer or at least the surface of the layer adjacent to the reagent layer or subbing layer. More particularly, the radiation diffusing and blocking layer can comprise any white or light coloured pigment incorporated into any suitable resin or polymeric material which is compatible with the substrate or support material on one hand and with the reagent layer or subbing layer on the other hand. If polymeric in nature, the radiation diffusing and blocking layer the be a homopolymer or a copolymer.
  • the radiation diffusing and blocking layer are between 0.0005 and 0.05 cm in thickness.
  • radiation impermeable pigments which can be used include titanium dioxide, barium sulfate, zinc oxide, magnesium oxide and zirconium dioxide. Normally, the upper limit on the amount pigment present will be less than 40% solids content and lower limit will be above 2% solids content.
  • suitable resins which can be employed for the radiation diffusing and blocking layer are anhydride resins which include methyl vinyl ether-maleic anhydride copolymer; ethylene-maleic anhydride copolymer; 1-octadecene-maleic anhydride copolymer; octadecyl vinyl ether-maleic anhydride copolymer and styrene-maleic anhydride copolymer.
  • Examples of other polymeric materials which can be used for the radiation diffusing and blocking layer are polyvinyl acetate, polyvinyl methacrylate, polybutadiene, polychoroprene and polyvinylpyrrolidone.
  • homogeneous specific binding assay techniques are based on the special interaction between (1) a conjugate of a binding component and a label and (2) a binding partner to the binding component in the conjugate, whereby a characteristic of the label is different when the label conjugate is bound by the binding partner compared to when such conjugate is not so bound.
  • the affected characteristic of the label may be of any measurable nature, for instance, a chemical or physical quality of the label.
  • the affected characteristic is a chemical reactivity in a predetermined reaction which involves the formation or breaking of chemical bonds, covalent or noncovalent.
  • the affected characteristic is a physical characteristic of the label which can be measured without chemical reaction.
  • the present test device will incorporate homogeneous specific binding assay reagents which interact with the ligand in or its binding capacity of the sample in an immunochemical manner. That is, there will be an antigen-antibody or hapten-antibody relationship between reagents and/or the ligand or its binding capacity in the sample.
  • immunoassays therefore are termed immunoassays and the special interaction between the label conjugate and its binding partner is an immunochemical binding.
  • the binding component of the label conjugate is an antigen, hapten or antibody (or a fragment thereof) and the binding partner is its corresponding immunochemical binding partner.
  • binding interactions between the label conjugate and the binding partner serve as the basis of homogeneous specific binding assays, including the binding interactions between hormones, vitamins, metabolites, and pharmo- cological agents, and their respective receptors and binding substances.
  • the reagents for the homogeneous specific binding assay technique comprise, in the usual case, (1) a label conjugate composed of the ligand, or a binding analog thereof, chemically coupled to the label, (2) a binding partner for the ligand, e.g., an antibody or fragment thereof or a natural receptor protein, and (3) any ancillary reagents necessary for measuring the labeling substance in the label conjugate.
  • a binding partner for the ligand e.g., an antibody or fragment thereof or a natural receptor protein
  • any ancillary reagents necessary for measuring the labeling substance in the label conjugate e.g., an antibody or fragment thereof or a natural receptor protein
  • the distribution of the label between the bound-species and the free-species will therefore determine the magnitude of the detectable response from the label which in turn will be a function of the presence of the ligand.
  • Another scheme for determining a ligand is presented where the label conjugate is composed of a labeled binding partner of the ligand and upon binding to the ligand the label is affected in terms of its detectable response.
  • the label conjugate will be composed of the ligand, or a binding analog thereof, chemically coupled to the label whereby the capacity of the sample to bind the label conjugate, such as due to the presence of a binding partner of the ligand in the sample, determines the effect made on the detectable signal from the label.
  • the label is a prosthetic group of an enzyme
  • the ability of a catalytically inactive apoenzyme to combine with the prosthetic group label to form an active enzyme (holoenzyme) is affected by binding of the label conjugate with its binding partner.
  • Resulting holoenzyme activity is measurable by conventional detectant systems to yield an ultimate detectable signal. Assay systems of this type are described in British Patent No. 2,023,607.
  • a particularly preferred prosthetic group- labeled assay scheme employs flavin adenine dinucleotide (FAD) as the label and apoglucose oxidase as the apoenzyme. Resulting glucose oxidase activity is measurable by a colorimetric detectant system comprising glucose, peroxidase, and an indicator system which produces a color change in response to hydrogen peroxide.
  • FAD flavin adenine dinucleotide
  • the FAD-label conjugate is preferably of the formula: wherein Riboflavin( ⁇ Phos) ⁇ 2 Ribose represents the riboflavin-pyrophosphate-ribose residue in FAD, R is a linking group, and L is the binding component, e.g., the ligand or analog thereof.
  • R is a linking group
  • L is the binding component, e.g., the ligand or analog thereof.
  • An example of such a conjugate is the FAD-theophylline conjugate disclosed in European Patent Application No. 81108681 (EP-A-0 051 213).
  • the label is selected so that the label conjugate is a substrate for an enzyme and the ability of the enzyme to act on the substrate-label conjugate is affected, either in a positive or negative sense, by binding of the label conjugate with its binding partner.
  • Action of the enzyme on the substrate-label conjugate produces a product that is distinguishable in some feature, usually a chemical or physical feature such as chemical reactivity in an indicator reaction or such as a photometric character, e.g., fluorescence or light absorption (color).
  • Assay systems of this type are described in DE-A-2,618,511 and US Patent No. 4,279,992 and in Anal. Chem. 48:1933 (1976), Anal. Biochem. 77:55 (1977) and Clin. Chem. 23:1402 (1977).
  • a particularly preferred substrate-labeled assay scheme employs a label conjugate of the structure wherein R is a linking group and L is the binding component, e.g., the ligand or analog thereof, whereby the ability of the enzyme (3-galactosidase to cleave the conjugate yielding a product distinguishable by its fluorescence is inhibited by binding of the conjugate with its binding partner.
  • the label conjugate in this system is composed, in its label portion, of a coenzyme-active functionality, and the ability of such coenzyme label to participate in an enzymatic reaction is affected by binding of the label conjugate with its binding partner.
  • the rate of the resulting enzymatic reaction is measurable by conventional detectant systems to yield an ultimately detectable signal. Assay systems of this type are described in DE-A-2,618,511; and in Anal. Biochem. 27:271 (1976), Anal. Biochem. 72:283 (1976) and Anal. Biochem, 76:95 (1976).
  • the label conjugate in this system is composed, in its label portion, of an enzyme modulating functionality such as an enzyme inhibitor or stimulator, and the ability of such modulator label to modulate the activity of an enzyme is affected by binding of the label conjugate with its binding partner.
  • the rate of the resulting enzymatic reaction is measurable by conventional detectant systems to yield an ultimately detectable signal. Assay systems of this type are described in commonly owned U.S. Patent No. 4,134,792.
  • the label is an enzyme and the activity of the enzyme label is affected by binding of the label conjugate with its binding partner. Resulting enzyme activity is measurable by conventional detectant systems to yield an ultimately detectable signal. Assay systems of this type are described in U.S. Patents Nos. 3,817,837 and 4,043,872.
  • the label conjugate in this system is composed, in its label portion, of a fluor the fluorescence of which is quenched in some measurable degree when the label conjugate is bound by its binding partner, usually a protein such as an antibody.
  • the fluorescent label is measured directly, with its fluorescence being the detectable signal. Assay systems of this type are described in U.S. Patent No. 4,160,016 and in J. Clin. Path. 30:526 (1977).
  • the label in this system is also a fluor; however, the affected characteristic is polarization of fluorescence due to binding of the label conjugate by its binding partner, usually a protein such as an antibody. Assay systems of this type are described in J. Exp. Med. 122:1029 (1965).
  • the label is again a fluor, however, the ability of the fluor label to be chemically excited to an energy state at which it fluoresces is affected by binding of the label conjugate with its binding partner. Chemical excitation of the label is usually accomplished by exposure of the fluor label to a high energy compound formed in situ. Assay systems of this type are described in DE-A-29 52 498.
  • the label conjugate comprises two epitopes, one of which participates in the immunological reaction with the ligand and antiligand antibody and the other of which is bindable by a second antibody, with the restriction that the two antibodies are hindered from binding to the label conjugate simultaneously.
  • the second epitope can be a fluor the fluorescence of which is quenched by the second antibody binding, or which may participate in an ancillary competitive binding reaction with a labeled form of the second epitope for binding to the second antibody.
  • Various detectant systems are possible in such a system as described in the aforementioned patents.
  • Related assay systems are described in U.S. Patents Nos. 4,130,462 and 4,161,515 and in British Patent * Spec. No. 1,560,852.
  • the label is one member of an energy transfer donor-acceptor pair and the binding partner is conjugated with the other of such pair.
  • the energy expression of the donor component of the pair is altered by trans- ferance to the acceptor component.
  • the donor is a fluor and the acceptor is a quencher therefor, which quencher may or may not be a fluor as well.
  • the detectable signal is fluorescence, but other detectant systems are possible also.
  • assay systems are described in U.S. Patents Nos. 3,996,345; 4,174,384; and 4,199,559 and in British Patent Spec. No. 2,018,424.
  • a method of preparing the reagent layer(s), radiation diffusing and blocking layer, or subbing layer, if the latter is used is also provided.
  • the layered devices of the present invention can be made by any suitable technique, such as printing, or by using any of the known film forming techniques.
  • the reagent layer actually comprises multiple layers
  • such layers can, if desired, be maintained in laminar relationship by adhesives which permit fluid passage between layers. Ordinarily, however, it is not necessary to employ adhesive to adhere one reagent layer to another.
  • the layer(s) can be preformed separately and laminated to form the overall element.
  • the material of the film layer(s) can be a composition comprising a plasticizer and a polymer suitable to impart dimensional stability. Layers prepared in such a manner are typically coated from solution or dispersion onto a surface from which the dried layer can be physically stripped.
  • a convenient method which can avoid problems of multiple stripping and lamination steps is to coat an initial layer on a stripping surface or a support, as desired, and thereafter to coat successive layers directly on those coated previously.
  • Such coating can be accomplished by hand, using a blade coating device, or by machine, using techniques such as dip or bead coating. If machine coating techniques are used, it is often possible to coat adjacent layers simultaneously using hopper coating techniques well known in the preparation of light sensitive photographic films and papers.
  • Blush polymer layers can be used as the film layer material.
  • the film is formed on a substrate by dissolving a polymer in a mixture of two liquids, one of which is of a lower boiling point and is a good solvent for the polymer and the other of which is of a higher boiling point and is a nonsolvent or at least a poor solvent for the polymer.
  • Such a polymer solution is then coated on the substrate, and dried under controlled conditions.
  • the lower boiling solvent evaporates more readily and the coating becomes enriched in the liquid which is a poor solvent or nonsolvent.
  • the polymer forms as a porous layer.
  • Many different polymers can be used, singly or in combination, for preparing porous blush polymer layers for use in this invention.
  • Typical examples include polycarbonates, polyamides, polyurethanes and cellulose esters, such as cellulose acetate.
  • a coating solution or dispersion including the matrix and incorporated active materials can be prepared, coated as discussed herein and dried to form a dimensionally stable layer.
  • any reagent layer and its degree of permeability are widely variable and depend on actual usage. Dry thicknesses of from 5 to 100 pm have been convenient, although more widely varying thickness may be preferable in certain circumstances. For example, if comparatively large amounts of interactive material, e.g., polymeric materials like enzymes, are required, it may be desirable to prepare slightly thicker layers.
  • surfactant materials such as anionic and nonionic surfactant materials
  • anionic and nonionic surfactant materials can, for example, enhance coatability of layer formulations and enhance the extent and range of wetting in layers that are not easily wetted by liquid samples in the absence of an aid such as a surfactant. It can also be desirable to include materials that can render nonactive in the analysis of choice, by chemical reaction or otherwise, materials potentially deleterious to such analysis.
  • This approach relates to a method for preparing the reagent layer of a homogeneous specific binding assay element for determining a ligand in or the ligand binding capacity of a liquid sample by incorporating a composition which includes a label conjugate, comprising a label component coupled to a ligand moiety or a specific binding analog thereof, and a reagent interreactive with the label conjugate.
  • the reagent can comprise, for example, a specific binding partner for the ligand or a specific binding partner for the ligand and a component which is interreactive with the label conjugate to cleave the label component from the ligand moiety or specific binding analog thereof.
  • the reagents can be applied in respective solutions which are incapable of substantial interreaction during preparation of the test device and thus do not react prematurely.
  • certain first reagents are incorporated with a layer using an aqueous dip.
  • a suitable organic solvent such as toluene, acetone, chloroform, methylene chloride, n-propanol and ethylene dichloride. This layer is set by allowing the organic solvent to evaporate.
  • An example of this preferred embodiment is a method for preparing a homogeneous specific binding assay device for determining a ligand in or the ligand binding capacity of a liquid sample by applying a composition which includes a f3-galactosyl-umbelliferone-ligand or ligand analog conjugate, ⁇ -galactosidase, and antiserum to the ligand which method comprises (a) applying f3-galactosidase and antiserum to the ligand in
  • a multizone reagent layer is prepared by (a) incorporating a first or overlaying zone with some, but less than all, of the reagents of the specific binding assay system used, (b) incorporating a second or underlaying zone with the remaining reagents, (c) setting, such as by drying, the individual zones, and (d) fixing them into laminar relationship with one another.
  • the first layer and second layer each have a pair of opposite surfaces.
  • One surface of the first layer is in laminar relationship with one surface of the second layer, sample being applied to the other surface of either of said layers.
  • Reference to a laminar relationship connotes the ability of a fluid, whether liquid or gaseous, to pass between superposed surfaces of such layers.
  • Such layers can be contiguous or separated by intervening layers. Any intervening layer should not prevent passage between all layers.
  • This approach consists of a procedure to incorporate and prevent reaction between incompatible reagents in a single layer analytical element.
  • a first group of reagents is applied by freeze drying or at elevated temperature and the treated layer is set.
  • the second group of reagents containing any which will react under ambient conditions with the first group, are applied and the element is rapidly frozen. Freezing prevents premature reaction and the subsequent removal of water by freeze drying prevents premature reaction when the layer is brought back to room temperature.
  • one group of reagents can be added in aqueous solution to a layer and dried.
  • the addition of a second group of reagents in aqueous solution is followed by rapid freezing and then freeze drying to remove water.
  • This procedure allows the incorporation of and prevents the interaction between some reagents which are only water soluble.
  • it avoids the use of organic solvents, certain of which may interact deleteriously with some reagents (e.g., enzymes).
  • the procedure permits formulation of elements utilizing homogeneous specific binding assay reagents in which all reagents are provided within a single layer element. an aqueous liquid and (b) then applying j3-galactosyl-umbelliferone-ligand or ligand analog conjugate in acetone.
  • the antibody and the label conjugate are kept separate until the introduction of the sample.
  • This embodiment of the described invention makes use of the reverse reaction and reequilibration with the ligand as shown by the equation below: where the amount of displaced label conjugate is related to the sample ligand concentration.
  • the advantage is that all reagent components can be combined in one incorporation medium to provide a system that requires only the addition of sample to be tested.
  • this approach provides a method of preparing the reagent layer of a homogeneous specific binding assay device for determining a ligand in a liquid sample, which method comprises (a) forming a complex between a label conjugate, the conjugate comprising a label component coupled to the ligand or a specific binding analog thereof, and a specific binding partner for the ligand; and (b) applying the complex.
  • forming the complex can comprise associating the label conjugate and specific binding partner therefor and allowing the conjugate, the binding partner and the complex to reach a state of equilibrium.
  • the layers are prepared by incubating a given conjugate with its respective antisera for a short period, such as 15 minutes. Then, any additional reagents are added and the system allowed to incubate an additional period. The solution so formed is then allowed to set.
  • the integral analytical elements include a support.
  • the support can be opaque, translucent or transparent to light or other energy.
  • a support of choice for any particular element will be selected independently of the intended mode of signal detection.
  • Preferred supports include those of polystyrene or similar plastics.
  • fixing a surface of the support layer to a surface of ths radiation diffusing and blocking layer comprises forming the radiation diffusing and blocking layer on the surface of the support layer. In another embodiment, fixing a surface of the support layer to a surface of the radiation diffusing and blocking layer comprises forming the radiation diffusing and blocking layer and, thereafter, fixing a surface of the radiation diffusing and blocking layer so formed to a surface of the support layer.
  • detectable species refer to atoms, chemical groups (i.e., a portion of a molecule) or chemical compounds that are themselves directly or indirectly detectable and the term “detectable response”, and similar terms as used herein, refer to the detectable manifestation of the presence of such species.
  • electromagnetic radiation signals such as fluorescence, phos- phorescense, chemiluminescence, a change in light absorption, or reflectance in the visible spectrum thereby producing a visible color change, a change in light absorption or reflectance outside the visible range such as in the ultraviolet range or infrared range.
  • the phrase "detectable response”, as used herein is intended in its broadest sense.
  • detectable response is also meant to include any observable change in a system parameter, such as a change in or appearance of a reactant, observable precipitation of any component in the test sample or a change in any other parameter, whether it be in the reagent system or the test sample.
  • detectable responses include electrochemical responses and calorimetric responses.
  • the detectable response is one which can be observed through the senses directly or by use of ancillary detection means, such as a spectrophotometer, ultraviolet light- sensing equipment, fluorometer, spectrofluorometer, and other similar sensing means. Desirably, such detectability can be conveniently imparted to the full amount of detectable species without affecting the amount of diffusible product resulting from the analyte interactions which are the basis of the intended analysis.
  • the analytical result is obtained as a detectable change, it is measured, usually by passing the test element through a zone in which suitable apparatus for reflection, transmission or fluorescence photometry is provided.
  • Such apparatus serves to direct a beam of energy, such as light.
  • the light is then reflected from the element back to a detector.
  • the analytical result is detected in a region of the element totally within the region in which such result is produced.
  • Use of reflection spectrophometry can be advantageous in some situations as it effectively avoids optical interference from any residues, such as blood cells or urine sediment, which have been left on or in the layers of the element or from atypical urine colors.
  • Conventional techniques of fluorescence spectrophotometry can also be employed.
  • electromagnetic radiation in the range of from about 200 to about 900 nm has been found useful for such measurements, although any radiation to which the reagent layer(s) is permeable and which is capable of quantifying the product produced in the element can be used.
  • Various calibration techniques can be used to provide a control for the analysis. As one example, a sample of a standard solution of the ligand under assay can be applied adjacent to the area where the drop of sample is placed in order to permit the use of differential measurements in the analysis.
  • Devices A and B have the support layer between the radiation diffusing and blocking and reagent layers, device B having a multizone reagent layer.
  • the reagent layer is between the support and radiation diffusing and blocking layers.
  • Device D has the radiation diffusing and blocking layer between the reagent and support layers, and is exemplary of the invention.
  • Device A was prepared by casting a reagent layer film of an agarose composition on one surface of the support layer and a Ti0 2 pigmented composition on the other surface to form a radiation diffusing and blocking layer.
  • the agarose composition was formulated as follows: Triton is a trademark for a line of synthetic organic surface-active agents sold by Rohm & Haas, Philadelphia, PA. Bicine is N,N-bis-(2-hydroxyethyl)-glycine.
  • the Ti0 2 pigmented composition was formulated as follows:
  • the preparation of device B was identical to that of device A, as described above, with the addition that a polyvinylpyrrolidone (PVP) composition was cast as a film onto the surface of the agarose film opposite from the surface in contact with the support layer.
  • PVP polyvinylpyrrolidone
  • the PVP composition formed a film which combined with the agarose film to form a multizone reagent layer and was formulated as follows:
  • Device C was prepared by casting a reagent layer film of an agarose composition on one surface of the support layer.
  • the agarose composition was identical to that used in Devices A and B.
  • the same Ti0 2 composition used for Devices A and B was then used to cast a film on the surface of the agarose film opposite from the surface in contact with the support layer.
  • Device D was prepared by casting a Ti0 2 pigmented composition, identical to that used in the other devices, onto a support layer to form a radiation diffusing and blocking layer and, then, casting an agarose composition, identical to that used in the other devices, onto the surface of the agarose layer opposite that in contact with the support layer.
  • the devices prepared as described above were each inserted into a mechanical holder suitable for horizontal positioning of the device in a fluorometer.
  • a 30 microliter (pL) drop of a 7-hydroxycoumarin-3-carboxanilide (HCC) aqueous solutions was placed on each device just prior to placing that device into the fluorometer.
  • HCC solutions used was in the range from 0.2 to 2.0 micromolar (pM) concentrations.
  • the fluorometer had been adjusted to provide an excitation light source of 405 nanometers (nm) wavelength, which struck the surface of the element at a 90° angle from the plane of the surface, and to detect light emitted at a wavelength of 450 nm.
  • a front face measurement of fluorescence was made at a 90° angle from the plane of the element surface. The fluorescence of each sample was then measured.
  • the data obtained using Devices A and B is represented in both Figs. 1 and 2 by the curve drawn in dashed line.
  • the data obtained using Devices C and D is represented in both Figs. 1 and 2 by the curve drawn in solid line.
  • the data obtained using Device E is represented in both Figs. 1 and 2 by the curve drawn in phantom line.
  • Fig. 1 illustrates a plot of the range as a function of the HCC concentration for each system.
  • Whatman 31ET Device E
  • two distinct responses are observed for the Ti0 2 systems.
  • the preparations with the Ti0 2 beneath the support (Devices A and B), whether gel bond or polyester, show a lower range than those with the TiO 2 on top of the support (Devices C and D), whether gel bond or polyester, in contact with the HCC solutions.
  • Whatman 31ET (Device E) shows a better range than the Ti0 2 systems.
  • Theophylline [1,3-dimethylxanthine, c.f. The Merck Index, 9th ed., p. 1196. (1976)] is a drug useful in the management of asthma. In most patients, the therapeutic range of serum concentration lies between 10 and 20 micrograms per milliliter ( ⁇ g/ml) whereas toxicity almost invariably appears at blood levels over 35 pg/ml.
  • Celite is a trade mark for certain diatomaceous earth products.
  • the impregnated Celite was placed atop a column of 200 g of silica gel (E. Merck Co., Darmstadt, West Germany) made up in 9:1 (v:v) ethanol-1 molar aqueous triethylammonium bicarbonate.
  • the column was eluted with this solvent and 15 ml fractions were collected. Fractions 171 to 225 were combined and evaporated to give 500 mg of a white powder.
  • a reaction mixture was prepared containing 24 g of potassium hydroxide, 80 ml of water, 240 ml of methanol and 20 g (0.035 mmol) of ethyl 7-j3-galactosylcoumarin-3-carboxylate [Burd et al, Clin. Chem. 23:1402 (1977)].
  • the reaction was stirred at 50°C for 15 hours.
  • the methanol was removed under reduced pressure.
  • the concentrated aqueous solution was acidified to pH 2.0 with concentrated hydrochloric acid.
  • the white precipitate was collected, washed with cold water, and recrystallized from hot water.
  • the crystals were collected, washed with acetone, and dried at 80°C for 1 hour. This gave 12 g of 7- ⁇ -galactosylcoumarin-3-carboxylic acid as white crystals, mp 250-255°C.
  • the filtrate was chromatographed on a 2.5 cm by 57 cm column of Sephadex LH-20 gel (Pharmacia Fine Chemicals, Piscataway, New Jersey), eluting with water and collecting 15 ml fractions.
  • Sephadex is a trade name for a hydrophilic, insoluble molecular-sieve chromatographic meduim, made by cross-linking dextran. Fractions 18 to 23 were combined, evaporated, and residue recrystallized from water to give 55 mg (2% yield) of the label conjugate (4) as a light yellow solid, mp 190-192°C.
  • Antiserum was collected from rabbits immunized with a theophylline immunogen conjugate prepared as described by Cook et al, Res. Comm. Chem. Path. Pharmaco/ 13:497-505 (1976).
  • the element prepared for use in these experiments had the configuration shown in Fig. 4.
  • the support layer 2 is of polystyrene.
  • the impermeable radiation diffusing and blocking layer 4 is of a composition having the following formulation: This composition was cast onto the polystyrene support layer 2 to a wet thickness of 0.05 cm (0.02 inches) and allowed to dry at room temperature.
  • a gelatin subbing layer 6 adheres to the surface of the impermeable radiation diffusing and blocking layer 4 and forms one zone of the multizone reagent layer 12.
  • the subbing layer is of a composition having the following formulation: This composition was cast onto layer 4 to a wet thickness of 0.013 cm (0.005 inches) and dried at 37° Centigrade (C).
  • An antibody and enzyme containing layer 8 was prepared of a composition having the following formulation: Agarose LGT is an agarose material having a low grilling temperature of below 40°C and is sold by the Research Products Division of Miles Laboratories, Inc., P.O. Box 2000, Elkhart, Indiana 46515. This composition was cast onto layer 6 to a wet thickness of 0.05 cm (0.02 inches) and dried at 37°C. It is the intermediate zone of the multizone reagent layer 12.
  • a conjugate containing layer 10 was prepared of a composition having the following formulation: This composition was cast onto layer 8 to a wet thickness of 0.005 inches and dried at room temperature. It is the uppermost zone of the multizone reagent layer 12. The exposed upper surface of layer 10 is the surface to which sample is applied and from which readings are taken.
  • the fluorescence generated at room temperature was measured for a duration of five minutes in a fluorometer equipped with a mechanical holder suitable for horizontally positioning the analytical element.
  • the fluorometer had been adjusted to provide an excitation light source at 405 nm, which struck the surface at 90° and to detect light emitted at a wavelength of 450 nm.
  • a front face measurement of fluorescence was made at a 90° angle from the pad.
  • the concentration ranges assayed were as follows: The dose response range checked covers the therapeutic range.
  • Fig. 5 The data obtained by the above-described procedure is graphically illustrated by Fig. 5.
  • the fluorescence (ordinate) is expressed in terms of millivolts (mv). A millivolt is one thousandth of a volt.
  • Fig. 6, was prepared from the data shown in Fig. 5 at the three (3) minute point. (The ordinate represents relative fluorescene).

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Claims (8)

1. Elément analytique multicouche destiné à détecter un ligand dans un échantillon liquide ou à détecter la capacité de fixation d'un ligand d'un échantillon liquide, du type ayant une couche réactive renfermant des réactifs qui sont sensibles au ligand présent dans l'échantillon ou à la capacité de fixation d'un ligand de l'échantillon pour produire une réponse détectable, une couche de diffusion et de blocage des radiations, et une couche de support, caractérisé en ce que la couche de diffusion et de blocage des radiations est (a) interposée entre la couche réactive et la couche de support; (b) imperméable au ligand, aux réactifs présents dans la couche réactive et aux produits de leur interréaction; (c) inerte envers le ligand, les réactifs présents dans la couche réactive et les produits de leur interréaction; (d) d'une épaisseur comprise entre 0,0005 et 0,05 cm; et (e) opaque, et renferme un pigment imperméable aux radiations.
2. Elément analytique multicouche suivant la revendication 1, caractérisé en ce que la couche de diffusion et de blocage des radiations constitue une résine d'anhydride.
3. Elément analytique multicouche suivant la revendication 1 ou 2, caractérisé en ce que la couche réactive comprend des réactifs pour un système homogène d'analyse par liaison spécifique qui produit une réponse détectable qui est une fonction de la présence du ligand dans l'échantillon ou de la capacité de fixation du ligand de l'échantillon.
4. Elément analytique multicouche suivant la revendication 3, caractérisé en ce que le système homogène d'analyse par liaison spécifique comprend un indicateur qui participe à une réaction enzymatique.
5. Element analytique multicouche suivant l'une quelconque des revendications 1 à 4, caractérisé en ce que la couche réactive comprend: une composition réactive renfermant
(i) un anticorps qui fixe le ligand
(ii) un conjugué du ligand ou d'un analogue de liaison du ligand, et d'un indicateur, et
(iii) un système détecteur qui est capable d'une interréaction avec l'indicateur pour produire une réponse détectable qui est différente lorsque le conjugué d'indicateur est lié par l'anticorps, comparativement au cas où il n'est pas lié de la sorte,

en sortie que la réponse détectable est une fonction de la présence du ligand dans l'échantillon liquide.
6. Elément analytique multicouche suivant les revendications 3 à 5, caractérisé en ce que:
(1) la couche réactive comprend:
(a) des réactifs pour un système homogène d'analyse par liaison spécifique qui produit une réponse détectable qui est une fonction de la présence du ligand dans l'échantillon ou de la capacité de fixation du ligand de l'échantillon; et
(b) un support solide renfermant les réactifs; et
(2) la couche de diffusion et de blocage des radiations comprend un pigment imperméable aux radiations incorporé à une résine d'anhydride, cette couche étant:
(a) interposée entre la couche réactive et la couche de support;
(b) imperméable au ligand, aux réactifs de la couche réactive, et aux produits de leur interréaction; et
(c) inerte au ligand, aux réactifs de la couche réactive et aux produits de leur interréaction.
7. Procédé de préparation d'un élément analytique multicouche suivant les revendications 1 à 6, procédé qui comprend les étapes de:
(1) fixation d'une surface de la couche de support à une surface d'une couche de diffusion et de blocage des radiations qui est
(a) imperméable au ligand, aux réactifs de la couche réactive et aux produits de leur interréaction; et
(b) inerte au ligand, aux réactifs de la couche réactive et aux produits de leur interréaction; et
(2) fixation d'une surface de la couche réactive à la surface opposée de la couche de diffusion et de blocage des radiations.
8. Méthode de détection d'un ligand dans un échantillon liquide ou de la capacité de fixation d'un ligand d'un échantillon liquide, qui consiste à faire entrer l'échantillon en contact avec un élément analytique multicouche du type ayant une couche réactive renfermant des réactifs qui sont sensibles au ligand dans l'échantillon ou à la capacité de fixation d'un ligand de l'échantillon pour donner une réponse détectable, une couche de diffusion ou de blocage des radiations et une couche de support, caractérisée en ce qu'on utilise un élément analytique multicouche suivant l'une quelconque des revendications 1 à 6.
EP82105549A 1981-07-06 1982-06-24 Elément d'analyse à plusieurs couches; procédé pour sa préparation et son emploi dans des procédés analytiques Expired EP0069281B1 (fr)

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ES8308076A1 (es) 1983-08-01
DK156925C (da) 1990-03-12
DK156925B (da) 1989-10-16
EP0069281A1 (fr) 1983-01-12
IE821619L (en) 1983-01-06
FI74819B (fi) 1987-11-30
AU548963B2 (en) 1986-01-09
CA1178536A (fr) 1984-11-27
DE3266533D1 (en) 1985-10-31
AU8559882A (en) 1983-01-13
FI74819C (fi) 1988-03-10
DK301782A (da) 1983-01-07
GR77252B (fr) 1984-09-11
ES513750A0 (es) 1983-08-01
FI822364L (fi) 1983-01-07
IL66210A0 (en) 1982-11-30
FI822364A0 (fi) 1982-07-02
IE53772B1 (en) 1989-02-15
US4390343A (en) 1983-06-28

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